The techniques of molecular biology have found extensive use in the cloning and analysis of DNA molecules. The most commonly used methods for cloning a gene sequence involve the in vitro use of site-specific restriction endonucleases, and ligases. In brief, these methods rely upon the capacity of the "restriction endonucleases" to cleave double-stranded DNA in a manner that produces termini whose structure (i.e. 3' overhang, 5' overhang, or blunt end) and sequence are both well defined. Any such DNA molecule can then be joined to a suitably cleaved vector molecule (i.e. a nucleic acid molecule, typically double-stranded DNA, having specialized sequences which permit it to be replicated in a suitable host cell) through the action of a DNA ligase. The gene sequence may then be duplicated indefinitely by propagating the vector in a suitable host. Methods for performing such manipulations are well-known (see, for example, Perbal, B. A Practical Guide to Molecular Cloning, John Wiley & Sons, NY, (1984), pp. 208-216; Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982); Old, R.W. et al., In: Principles of Gene Manipulation, 2nd. Ed., University of California Press, Los Angeles, (1981), all herein incorporated by reference).
Depending upon the size and characteristics of the desired target molecule, any one of the three different types of vectors--plasmids, bacteriophage, or cosmids--can be employed (See, generally, Watson, J.D., In: Molecular Biology of the Gene, 4th Ed., W.A. Benjamin, Inc., Menlo Park, CA (1987), which reference is incorporated herein by reference).
The use of plasmids in cloning is quite well known (see, for example, Cohen et al., U.S. Pat. No. 4,237,224; Itakura, U.S. Pat. No. 4,356,270; Fraley, R.T. et al., PCT Application WO 84/02919; etc.). In general, the most pronounced deficiency of plasmid vectors is the relatively small amount (up to about 5-10 kb) of DNA which can be cloned into them. In general, the larger the size of the target molecule, the lower the efficiency of plasmid transformation.
Bacteriophage vectors, and particularly vectors engineered from the bacteriophage .lambda. have been extensively used as cloning vehicles (see generally, The Bacteriophage Lambda, (Hershey, A.D., ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1971), and Lambda II, (Hendrix, R. et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 175-209 (1983), Maniatis, T., et al. (In: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982), all herein incorporated by reference). In general, bacteriophage vectors have the advantage that they can be used to clone gene sequences of up to approximately 23 kb.
Cosmids are vectors which have been specifically designed to facilitate the cloning of large DNA molecules. The essential components of a cosmid vector are (1) a drug-resistance marker; (2) a plasmid origin of replication; (3) one or more unique cloning sites that are recognized by a restriction endonuclease; (4) a cos site of bacteriophage .lambda.. Cosmid cloning methods are described, for example, by Maniatis, T., et al, (In: Molecular cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Cosmids vectors can be used to clone 40-50 kb of target molecule. They are thus highly useful in the cloning and analysis of eukaryotic DNA, and especially mammalian genomes. Despite the ease with which in vitro cloning manipulations are often accomplished, serious impediments often limit their application in specific instances.
In vitro cloning may result in genetic rearrangements, deletions or insertions occurring in the desired gene sequence. It often requires the substantial prior purification of gene fragments. Such purification may be impeded if the desired gene fragment does not amplify at the same rate, or with the same fidelity, as other fragments. These concerns are particularly important with regard to the cloning of large DNA fragments, such as those containing human genes and gene families.
Moreover, the capacity to move target molecules from one vector to another in vitro is often limited by the availability or suitability of restriction sites. For example, in order to complete a desired cloning manipulation, it may be necessary to remove a particular enzyme that was needed in an initial cloning step. Such removal would necessitate a time-consuming purification of the cloning intermediate. Thus, a method for effecting the transfer of a target molecule from one vector to another that would not require such intermediate processing would be desirable. The present invention provides such a method and vector molecules for use therein.